Problem

The vaginal microbiome is unique and dynamic – it changes every day and differs between each person. Current diagnostic tools fail to capture this complexity, which presents problems because patients lack crucial data which is necessary for informed decision-making regarding their vaginal health.

Background

To understand our solution, you first need to understand some background information about diagnostic tools and the vaginal microbiome.

1. Microbes as Diagnostic Tools

Diagnostic tools in general have two main functions:

1. To detect stimuli from the environment
2. To communicate that information in a meaningful way

Plasmids are one such device that can be engineered to detect specific signals. Plasmids are circular pieces of double-stranded DNA that exist inside bacteria such as E. coli. Using plasmids as biosensors means that we are manipulating naturally existing biology to detect biological signals – kinda cool right?

In plasmid genetic circuits, detection can be achieved by creating a “place” for the molecule of interest to bind to. This special “place” must be able to interact with the molecule of interest, and then cause a measurable reaction.

Promoters are the answer to this problem. They are short sequences of DNA that enable the following DNA to be transcribed. Promoters can also be turned on or off – like an oven dial – depending on the surrounding factors. When the molecule of interest is present, it will bind to the promoter, and then the DNA will be transcribed!

After the promoter, there must be some way for the following DNA to send a meaningful signal. There are many different options for what this reporter signal can be, but we chose to use a fluorescent protein.

To recap, the most basic genetic circuit that accomplishes the two goals of diagnostic tools is as follows:

1. Promoter: (detector) “turns on” in the presence of specific molecules
2. Fluorescent protein: (reporter) produces a measurable signal that to communicate promoter binding

2. Bacterial Vaginosis

Bacterial vaginosis (BV) is defined as a collection of symptoms caused by an imbalance between bacteria in the vaginal microbiome, namely Gardnerella vaginalis and Lactobacillus spp. In a healthy microbiome, Lactobacillus is dominant. However in a BV state, Gardnerella dominates the microbiome. Gardnerella abundance in the vaginal microbiome has been shown to be correlated to clinically diagnosed vaginal odor [2] which is caused by the presence of metabolites called biogenic amines. One biogenic amine that has been linked to BV is putrescine [3, 4].

Solution

Our solution builds off a basic detector-reporter genetic circuit, with a few extra steps to ensure accurate measurements.

Putrescine-responsive genetic circuit

We used a promoter that is sensitive to putrescine, designed by Selim et al. [5] The amount of putrescine that binds to the promoter can be used to determine the relative abundance of putrescine in the sample. Putrescine alleviates the promoter repression, which enables DNA transcription and optical signal production.

Our design is based on a protein PuuR, which is sensitive to putrescine.

With no putrescine present, the PuuR molecule binds to the custom promoter. In the bound state the promoter is “turned off”, so the downstream signal is suppressed.

If there is putrescine present, the PuuR molecule binds to putrescine instead of the promoter, thus removing the “repressor” on the promoter. This enables downstream transcription.

Downstream of the custom promoter there is a green fluorescent protein (GFP). When the promoter is turned on in the presence of putrescine, GFP will be produced.

To produce the PuuR repressor molecule, we designed a secondary circuit on the same plasmid. This circuit begins with a constitutive (always on) promoter, followed by the PuuR coding sequence. After this, we have a red fluorescent protein (RFP). This serves to verify that the PuuR protein is being produced. Also, since this RFP should always be on, it can give us a measure to quantify the GFP amount against.

Constitutively Expressed Genetic Circuit

This circuit helps to characterize our putrescine-responsive genetic circuit. The only change with this circuit is that the putrescine-inducible promoter is exchanged for a second T7 promoter. This circuit should not respond to putrescine, and instead serve to provide baseline information to characterize the putrescine-inducible promoter.